07/08/09

PROCEDURE FOR THE REMOVAL OF SDS FROM PROTEIN SOLUTIONS

This protocol is based on the method of Wessel & Flugge, Anal. Biochem. 138, 141-143 (1984), and describes
protein precipitation from detergent-containing solutions. Recommended initial sample volume is 100 µL (if lower,
make up to 100 µL with water). Approximately 1 µg protein is required to visualize the pellet, though greater than
5 µg is desired. To become familiar with the protocol, it is recommended to first attempt precipitation of 50 µg
protein (pure standard, or a mixture). The protocol anticipates protein yields of 50-90%, and a ~1000-fold
reduction of SDS. A simple SDS colorimetric assay is described by Arand et al, Anal. Biochem. 207, 73-75 (1992).

Beginning with 100 µL of protein sample in a 1.5 mL eppendorf vial:

1. Add 400 µL methanol to the sample; vortex briefly (1-2 sec). Note: volume ratios must be maintained
throughout the protocol, thus for example beginning with 150 µL protein sample, add 600 µL methanol

2. Add 100 µL chloroform to the sample; vortex briefly. Note: all solvents are miscible to this point
3. Add 300 µL of deionized water; vortex briefly. Note: the solution will immediately become cloudy, as
methanol/water and chloroform are no longer miscible at these ratios.

4. Centrifuge the sample for 10-15 min near top speed on a benchtop centrifuge (~13,000 rpm or
~9000g). Note: chloroform will settle as bottom layer, protein at the interface, and methanol/water as the top
layer which contains most of the SDS. A cloudy layer often gradually forms (~1-2 min) at the interface.

5. Allow the vial to sit on the benchtop for ~15 min, until the cloudy layer disappears and a clear
interface is visible. Note: cloudy layer results from solvent mixing - it is not related to the protein itself. If
sufficient protein is present (>5 µg), the pellet will be visible at the interface once the cloudy layer settles.

6. With the vial at a ~45° angle, gently pipette the top layer, leaving approximately 100 µL of this top
layer in the vial. Note: pipet slowly, working from the top down, being careful not to disturb the protein pellet.
7. Centrifuge briefly vial (~10 seconds) at high speed to force down any liquid droplets which may
cling to the sides of the vial.
8. With vial at a ~45o angle, gently pipet the top layer of solution until ~20-50 µL solution remains.
Note: use a P100 pipet. It is best to stop once the top layer forms a bead in the vial

9. Slowly add 400 µL methanol to the vial. Note: while miscible, the methanol will simply float on top of the
chloroform layer until it is mixed. The protein pellet should be clearly visible as a small white flake at the
interface of these two layers.

10. Gently tap the vial 3-5 times, forcing the two layers to swirl together. Note: the goal is to mix the
solvents together, without breaking the pellet into several smaller pieces. This swirling step provides the best
opportunity to visualize the protein pellet (even if it was not visible previously)

11. Centrifuge the sample near top speed (~13,000 rpm) for 10-15 minutes. Note: pellet should settle to
the bottom of vial

12. With the vial at a ~45° angle, gently remove the solution, leaving ~100 µL in the vial. Briefly
centrifuge (10 seconds) to force any remaining liquid to the bottom of the vial, then carefully
remove the solution until ~10 – 50 µL remains in the vial. Note: leave behind larger volume of
solution if the pellet is difficult to visualize

13. Gently add 400 µL methanol to the vial. Do not mix at this point. Centrifuge & discard supernatant
as described in steps 11-12. Note: an additional washing step can be incorporated here
14. Place the open vial in a dust-free hood to allow any remaining liquid to evaporate. Note: protein
pellet can be resolubilized with 8M Urea, or MS-compatible surfactants (eg PPS silent surfactant)

Dr. Alan Doucette, Associate Professor of Chemistry, Dalhousie University , Halifax, Canada • alan.doucette@dal.ca
 07/08/09

A B

Shown in panels A & B is the cloudy layer which forms following the first
centrifugation (step 4). This layer is formed as a result of solvent mixing, and should
not be confused with the protein pellet.

C D

The pellet (from 50 µg) is visible at the interface in panel C (step 6), following partial
removal of the upper solvent layer. Continue to remove this upper layer until the
solvent beads (panel D).

E F

Following addition of methanol (step 9), the pellet is visible as a single white flake
(Panel E). When swirling the solvents, avoid breaking up this pellet. Panel F shows
the final pellet, prior to evaporating any remaining solvent (step 14).

Dr. Alan Doucette, Associate Professor of Chemistry, Dalhousie University , Halifax, Canada • alan.doucette@dal.ca